Tissue engineering approaches have been explored as a means to create grafts for ligament reconstruction procedures without potential donor-site morbidity. Our long-term goal is the creation of a tissue-engineered bone-ligament-bone graft that fully reproduces the tissue architecture found at the insertions in vivo. We believe that both extracellular matrix (ECM) alignment and controlled ECM heterogeneity are crucial elements to the long-term success of such an autologous implant. However, use of patient-derived marrow stromal cells (MSCs) to produce grafts with these characteristics is hampered by a lack of clear understanding of what occurs near the ligament-bone interface to direct alignment and differentiation of these cells. Therefore, as a first step toward our long-term goal, the objective of this application is to use a unique system combining a novel layered, enzyme-sensitive hydrogel carrier and precise control of macroscopic loading parameters to determine how two important characteristics of the extracellular environment, physicochemical properties of the surroundings and co-culture with osteoblasts, influence alignment and phenotypic expression by MSCs. The central hypothesis of this proposal is that, under cyclic tension, expression of the fibroblastic phenotype by MSCs can be modulated in a predictable manner by altering 1) physicochemical characteristics of the microenvironment and 2) the presence of nearby osteoblasts. Our overall objective will be accomplished by testing our central hypothesis in the following two specific aims: 1) Determine the effect of biochemical properties (adhesive ligand concentration) and physical properties (enzymatic degradation of the polymeric network) of the hydrogel microenvironment on cellular alignment and the extent of fibroblast phenotypic expression by encapsulated rabbit MSCs exposed to cyclic tensile loading over 21 days. 2) Determine the effect of the presence of osteoblasts on the timing and extent of fibroblastic/fibrochondrocytic differentiation by encapsulated rabbit MSCs under cyclic tensile loading over 21 days. The proposed work is innovative because the combination of a novel, well-defined three- dimensional cellular environment, including the laminated structures allowing for the co-culture of MSCs and osteoblasts, and the precise control of macroscopic mechanical loading provide a unique platform for controlled study of the influences on MSC differentiation near the bone-ligament interface. Completion of these studies is expected to distinguish the effects of the physicochemical properties of the microenvironment and the interplay with neighboring cells on the fibroblastic differentiation of MSCs. Such key information will direct the design of future strategies for production of patient-specific tissue-engineered grafts to replace damaged ligaments and restore full joint function. PUBLIC HEALTH RELEVANCE: This proposal examines the effects of 1) chemical properties of the microenvironment and 2) interplay with neighboring cells on fibroblastic differentiation of marrow stromal cells in order to create a tissue-engineered bone-ligament-bone graft that reproduces the tissue architecture found at ligament-bone insertions in vivo.